The goal of this project is to define the function of the basic helix-loop-helix transcription factor Hand2 during mammalian lower jaw development. Our hypothesis is that Hand2 contributes to the development of most lower jaw structures by establishing a distal morphogenetic domain in the mandibular arch. Within this boundary, Hand2 confines genes involved in mandibular pharyngeal arch development while also repressing the expression of genes normally observed outside this domain. Further, we hypothesize that Hand2 expression and function is at least partially regulated by Twist1 through both transcriptional repression and genetic interactions. In both mouse and zebrafish, Hand2 is expressed in cranial neural crest cell (NCC)-derived cells within the mandibular pharyngeal arch, from which bone and cartilage of the lower jaw arise. Targeted inactivation of the Hand2 gene in mice results in early embryonic lethality (by E10.5). However, hand2-mutant zebrafish are viable for five days;in these mutants, extensive malformations in craniofacial cartilages are accompanied by positive and negative changes in gene expression. We have shown in term mouse embryos that Hand2-daughter cells compose most structures derived from the mandibular arch. In our Preliminary Data, we show that loss of Hand2 in NCCs resultst in craniofacial defects indicative of NCC mispatterning. In this proposal, we will address the role of Hand2 in facial morphogenesis in three Specific Aims. In Aim 1, we will define the cellular and molecular changes in facial development following conditional inactivation of the Hand2 gene in cranial neural crest cells. In Aim 2, we will use hand2 zebrafish mutants to examine whether Hand2 acts as both a transcriptional activator and repressor and use Hand2 conditional mouse mutants to identify targets of Hand2 action. In Aim 3, we will define the role of Twist1 in the Hand2 mandibular arch domain and determine whether Twist1 and Hand2 interact genetically within this domain. By coupling our cellular and molecular analysis of Hand2 conditional knockout mice with our functional analysis of Hand2 action in mouse and zebrafish, we expect to uncover novel regulatory mechanisms governing fate and identity of NCCs that ultimately lead to facial development and whose disruption can lead to human facial birth defects, including micrognathia. PUBLIC HEALTH RELEVANCE: Craniofacial birth defect syndromes occur in 1 out of every 250 live births and represent a large financial and social burden within our society. While numerous mouse developmental genetics studies have elucidated the basis of some of these syndromes, the cause of many more remains unknown. The phenotype of the hand2 mutant zebrafish indicates that Hand2 may be involved in establishing or maintaining a developmental domain necessary for mandibular arch development, though early lethality prevents analysis of the role of Hand2 in later arch patterning events and bone formation. Our study will directly address the function and regulation of Hand2 during lower jaw development using a combination of model systems. Together, our studies will help define how regional developmental domains necessary for facial development are established and how different domains may antagonize each other to produce a final facial plan. Such knowledge can be subsequently combined using a systems biology approach to build a more comprehensive "gene map" involved in facial formation, which could lead to significant advances in future tissue engineering approaches to treat human craniofacial anomalies.